SKIN PENETRATION BY COPPER-PEPTIDE COMPLEXES 61 In the literature we may find that GSH has an activity of reducing free radicals and peroxides that are responsible for tyrosinase activation, melanin formation, and modu­ lation of the depigmenting abilities of melanocytotoxic agents. This leads to the skin lightening effect of GSH application and a possibility of its usage in the treatment of pigmentary disorders (17-20). Another important issue in skin protectioning is the anti-UV (UVA and UVB) radiation activity of the cosmetic ingredient. Glutathione is one of the ingredients that may play such a role (21-24). Due to the lack of data concerning the transport of the peptides and their complexes through the skin, we focused our research on this subject. The main goals of our experiments were to prove the ability of copper tripeptide complexes to penetrate the skin, to determine the permeability coefficient for these compounds, and to establish the form of the compound that actually penetrates through the membrane. Our previous study (25) proved that cooper peptides can migrate through the model lipophilic mem­ brane from an aqueous solution (25), which made us continue the investigation of the transport of copper peptide complexes, but this time through an emulsion. Since most of the cosmetic formulae used as a source of active ingredients, like peptides and their complexes, are O/W emulsions, we used them in our investigations. The in vitro penetration process was studied in the model system, a Franz diffusion cell (26-28) with a liposome membrane, where liquid crystalline systems of physicochemical prop­ erties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier (29-32). MATERIALS AND METHODS TYPES OF APPARATUS The absorption spectra were recorded using a SPECOL 11 spectrophotometer (Zeiss, Jena, Germany) with 5-mm glass cells. The pH measurements were carried out using an Elmetron ES24 pH meter (Poland). The reversed-phase liquid chromatographic experiment (RPLC) was performed by a Perkin Elmer binary LC 250 computer-controlled pump (Norwalk, CT) and a Rheodyne model 7125 injection vial with injection loops (20 µl) (Cotati, Rheodyne, CA) with a Perkin Elmer model LC-95 UV/Vis spectrophotometric detector. Peptides were sepa­ rated on the Hypersil BOS C18 analytical column (4.0 x 125 mm) (Agilent Technolo­ gies, Wilmington, NC). The acquisition and handling of the data were carried out with a 1020 LC Plus (Perkin Elmer) computer program. The copper peptide complexes were characterized by an ESI mass spectrometer, LC-MSD ll00 (Agilent) with a quadrupole mass analyzer (HP7500A). REAGENTS • A stock Cu(II) solution, (1 mg/ml- 1 ) was obtained by dissolution of copper(II) chlo­ ride dehydrate (POCH, Gliwice, Poland) in water. • A GHK-Cu solution (0.01 M) was prepared by dissolution of Prezatide copper acetate (GHK-Cu) (ProCyte Corporation, USA) in water. • A GSH stock solution (l0mg/ml) was prepared by dissolution of glutathione (Sigma-

62 JOURNAL OF COSMETIC SCIENCE Aldrich) in water. The solution was diluted in a calibrated flask. The complex of GSH with Cu was molar ratio 2:1 and 1:1. • A buffer solution (pH 7.4) was prepared by dissolving potassium phosphate (POCH, Gliwice, Poland), and its pH was adjusted to 7.4 by addition of di-sodium hydrogen phosphate dodecahydrate (POCH, Gliwice, Poland). The obtained solution was di­ luted to 1000 ml with demineralized water. • A 0.1 % biscyclohexanon-oxalyldihydrazone (cuprizon) (Fluka, Buchs, Switzerland) solution was prepared by dissolving 200 mg of cuprizon in 40 ml of hot 50% ethanol. This solution was diluted with ethanol to 200 ml. • A buffer solution (pH 10.0) was prepared by dissolving ammonium chloride (POCH, Gliwice, Poland) and was adjusted to 10.0 by addition of ammonium (POCH, Gli­ wice, Poland). The obtained solution was diluted to 1000 ml with demineralized water. • Trifluoroacetic acid (TFA) solution (0.15%) (Fluka, Buchs, Switzerland) was prepared by dissolving an appropriate amount in distilled water. The solution was diluted in a calibrated flask. • Components of the model emulsion: 8% glyceryl stearate (Cutina GMS) 20% hexyldecanol, hexyldecyl laurate (Cetiol PGL) 3% emulsifier-Ceteareth-20 (Eumul­ gin B2) 0.1 % methylchloroisothiazolinone, methylisothiazolinone (Kathan CG) and water-q.s. PREPARATION OF THE MEMBRANE The lipophilic membrane for modeling stratum corneum lipids was prepared by sand­ wiching 0.125 ml of liposomes (Cerasome) (Lipoid GmbH, Germany) composed of the horny layer lipids. The appropriately thick lipid layer was placed between two mem­ branes (Institute of Chemistry and Nuclear Technique, Poland) of polyester foil (radius, 12 mm diameter of pores, 0.4 micrometer thickness, 12 micrometers). The membrane was left for 24 hours to evaporate the water. EXPERIMENT AL In vitro membrane permeation experiments were performed using a Franz diffusion cell. The acceptor cell was filled with 15 ml of phosphate buffer (pH 7 .4). One gram of a 0/W emulsion containing copper complexes with peptides was placed in the donor cell. The available diffusion area between cells was 1. 77 cm 2 • The contents of the cells were stirred at 1000 rpm by a magnetic stirrer. During the 72 hours of experiments, the water from the emulsion was evaporated. The experiments were conducted at room tempera­ ture. Copper was determined spectrophotometrically at 600 nm. One milliliter of the solution from the acceptor cell (during 72 hours) was transferred into a 10-ml calibrated flask, and 2 ml of 0.1 % cuprizon and 2 ml of buffer solution (pH 10.0) were added. The mixture was diluted to 10 ml in a calibration flask, and the absorbance of the solution at 600 nm against a reagent blank was measured (33). The determination of the total amount of tripeptide in the acceptor cell was carried out by RPLC. A 1-ml sample was carried out from the acceptor cell. A 20-µl portion of this sample was injected onto the column. The flow rate of the eluent (0.15% TFA) was 0.7